Sentin functions, at least partially, independent of XMAP215^msps in S2 cells. (A) Immunoblotting to determine knockdown efficiency of Sentin and XMAP215^msps after RNAi. Sentin RNAi was very efficient, whereas ∼35% residual XMAP215^msps was present after RNAi. Tubulin, Aug5 (Dgt5), and the Coomassie staining were used as loading controls. (B) MT growth rate determined by tracing EB1-GFP signals in the kymograph after control (n = 94), Sentin (n = 94), XMAP215^msps (n = 47), and double Sentin/XMAP215^msps RNAi (n = 49). The error bars represent SEM. (C) The Sentin fragment that lacked the first 230 aa did not restore the normal MT growth rate, although it restored XMAP215^msps-GFP accumulation at the tip. Further truncation of the N terminus of Sentin did not rescue either defect. In this experiment, three independent stable cell lines expressing mRFP- or mCherry-tagged Sentin truncations and XMAP215^msps-GFP were used, and growing MT ends were identified by mRFP/mCherry signals. Plus-end accumulation of XMAP215^msps-GFP was visually determined after kymograph generation (51–121 MTs from two to seven cells). The kymographs and a still cell image of mRFP/mCherry and GFP signals after RNAi knockdown of endogenous Sentin are shown at the top. The XMAP215^msps-GFP accumulation frequency and MT growth rate relative to control RNAi for each cell line (±SEM) have been plotted below. (D, left) A monopolar spindle after double Sentin/Klp61F RNAi. A sum projection image of 11 z sections (separated by 0.5 µm) is shown, and the centrosome and astral MT ends are marked. Chromosomes were clustered in the lower side in this cell (not visible in this image). (right) The distance between a centrosome and each astral MT end was measured after sum projection, and the relative value to control RNAi cells has been plotted. Data have been provided for control (n = 187), Sentin (n = 322), XMAP215^msps (n = 169), and double Sentin/XMAP215^msps RNAi (n = 174) from 22 or 23 cells (±SEM). Bars: (horizontal) 5 µm; (vertical) 1 min.